Cellodextrin and laminaribiose ABC transporters in Clostridium thermocellum.
Clostridium thermocellum is an anaerobic thermophilic bacterium that grows efficiently on cellulosic biomass. This bacterium produces and secretes a highly active multienzyme complex, the cellulosome, that mediates the cell attachment to and hydrolysis of the crystalline cellulosic substrate. C. thermocellum can efficiently utilize only beta-1,3 and beta-1,4 glucans and prefers long cellodextrins. Since the bacterium can also produce ethanol, it is considered an attractive candidate for a consolidated fermentation process in which cellulose hydrolysis and ethanol fermentation occur in a single process. In this study, we have identified and characterized five sugar ABC transporter systems in C. thermocellum. The putative transporters were identified by sequence homology of the putative solute-binding lipoprotein to known sugar-binding proteins. Each of these systems is transcribed from a gene cluster, which includes an extracellular solute-binding protein, one or two integral membrane proteins, and, in most cases, an ATP-binding protein. The genes of the five solute-binding proteins were cloned, fused to His tags, overexpressed, and purified, and their abilities to interact with different sugars was examined by isothermal titration calorimetry. Three of the sugar-binding lipoproteins (CbpB to -D) interacted with different lengths of cellodextrins (G(2) to G(5)), with disassociation constants in the micromolar range. One protein, CbpA, binds only cellotriose (G(3)), while another protein, Lbp (laminaribiose-binding protein) interacts with laminaribiose. The sugar specificity of the different binding lipoproteins is consistent with the observed substrate preference of C. thermocellum, in which cellodextrins (G(3) to G(5)) are assimilated faster than cellobiose.
Nataf
Yakir
Y
Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel.
Yaron
Sima
S
Stahl
Frank
F
Lamed
Raphael
R
Bayer
Edward A
EA
Scheper
Thomas-Helmut
TH
Sonenshein
Abraham L
AL
Shoham
Yuval
Y
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001692:Biological Transport; D002151:Calorimetry; D002482:Cellulose / Q000031:analogs & derivatives* / Q000378:metabolism; D048013:Acetivibrio thermocellus / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D017931:DNA Primers; D003912:Dextrins / Q000378:metabolism*; D004187:Disaccharides / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D006094:Gram-Positive Bacteria / Q000378:metabolism; D007785:Lactose; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D010957:Plasmids; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D014158:Transcription, Genetic; D047071:beta-Glucans / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D017931:DNA Primers; D003912:Dextrins; D004187:Disaccharides; D009097:Multienzyme Complexes; D012329:RNA, Bacterial; D047071:beta-Glucans; C014637:laminaribiose; D002482:Cellulose; C068994:cellodextrin; D005947:Glucose; D007785:Lactose